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US6043058A - Preparation of cyclic depsipeptide compounds and a novel cyclic depsipeptide - Google Patents

Preparation of cyclic depsipeptide compounds and a novel cyclic depsipeptide Download PDF

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US6043058A
US6043058A US09/242,041 US24204199A US6043058A US 6043058 A US6043058 A US 6043058A US 24204199 A US24204199 A US 24204199A US 6043058 A US6043058 A US 6043058A
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strain
crystals
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Makoto Ohyama
Masaaki Takahashi
Yoshiya Shigematsu
Osamu Sakanaka
Yashushi Murai
Katsuharau Iinuma
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Maiji Seika Kaisha Ltd
Meiji Seika Kaisha Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D273/00—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/14—Nitrogen or oxygen as hetero atom and at least one other diverse hetero ring atom in the same ring

Definitions

  • This invention relates to a fermentative process for the preparation of PF 1022F substance and PF 1022H substance which are known cyclic depsipeptides, and for the preparation of PF 1022G substance which is a novel depsipeptide.
  • This invention further relates to PF 1022G substance as the novel depsipeptide.
  • PF 1022 substance (which may also be called as PF 1022A substance) as a product of a microorganism, and PF 1022 substance is classified under cyclic depsipeptides having an anthelmintic activity (refer to Japanese Patent Application First Publication Kokai Hei 3-35796, Japanese Patent No. 2608479, U.S. Pat. No.5,116,815 and European Patent Application First Publication No. 0382173A2).
  • This PF 1022 substance is the cyclic depsipeptide represented by the following formula (A): ##STR2## wherein Me stands for methyl group Me has the same meaning in the following descriptions given hereinafter.
  • PF 1022 substance is a cyclic depsipeptide constituted by L--N-methylleucine [(CH 3 ) 2 CHCH 2 CH(NHCH 3 )COOH] (Code: H--L--MeLeu--OH), D-lactic acid [CH 3 CH(OH)--COOH] (Code: H--D--Lac--OH) and D-phenyllactic acid [C 6 H 5 CH 2 CH(OH)COOH] (Code: H--D--PhLac--OH), which are bonded with each other through ester- and amido-bonds.
  • PF 1022 substance may also be represented by the following formula (B).
  • PF 1022B substance, PF 1022C substance and PF 1022D substance are PF 1022-related compounds having an anthelmintic activity (refer to Japanese Patent Application First Publication Kokai Hei 5-170749).
  • PF 1022E substance was found as a product of the microorganism (refer to Japanese Patent Application First Publication Kokai Hei 6-184126).
  • the microorganism used here is PF 1022 strain hereinafter described.
  • Examples 3 and 8 of the above PCT Internationally published specification WO94/19334 describe PF 1022-002 substance and PF 1022-202 substance, respectively, which were chemically synthesized by the present inventors.
  • PF 1022-002 substance is reported in another name of PF 1022F substance
  • PF 1022-202 substance is reported in another name of PF 1022H substance in PCT Internationally published specification WO97/11064 (published on Mar. 27, 1997) of PCT Application PCT/JP96/02730(with International filing date: Sep. 20, 1996).
  • the PCT Application PCT/JP96/02730 was filed with claiming a priority from Japanese Patent Application Hei 7-244051 filed on Sep. 22, 995 and is claiming a series of such novel derivatives of PF 1022 substance which are recently synthesized by the present inventors.
  • PF 1022H substance is a cyclic depsipeptide obtained through a chemical synthesis by the present inventors, and we have further found that this PF 1022H substance is also useful as a starting material for the preparation of a series of depsipeptide derivatives possessing an improved anthelmintic activity, if the functional group or groups of PF 1022H substance is or are chemically modified further.
  • the fermentative process for preparing the cyclic depsipeptides having such a complex cyclic skeleton as seen in the structural formula (A) above there are two routes, namely a route by chemical synthesis and another route by cultivation of a microorganism.
  • the fermentative process for preparing the cyclic depsipeptides by cultivation of microorganism shall leave the production of the intended substances of the complex structure to the actions of the microorganism used.
  • the fermentative process is generally advantageous in practice, in respect of the overall period of time required, labors and expenses and other points, and the fermentative process may be operated in a easier and more convenient way.
  • the PF 1022F substance and PF 1022H substance are requested to be prepared in a more convenient way by the cultivation of microorganism rather than by the chemical synthetic route. It is also requested that such novel cyclic depsipeptides which have not yet been disclosed in literature are prepared through such convenient, fermentative process by the cultivation of a microorganism, because such novel cyclic depsipeptides will have such possibilities that they possess an anthelmintic activity or some other pharmaceutically useful activities, and that they are utilizable as intermediate materials for chemically synthesizing some other derivatives possessing some useful activities.
  • One of the objects of this invention is, therefore, to provide a novel process for the preparation of the PF 1022F substance and PF 1022H substance by the cultivation of a microorganism.
  • Another object of this invention is to provide novel, useful cyclic depsipeptides by the cultivation of a microorganism.
  • PF 1022G substance a novel cyclic depsipeptide is also produced in the same culture broth of the PF 1022 strain, and we have designated this novel substance as PF 1022G substance. Also, we have succeeded in recovering the PF 1022F substance, PF 1022H substance and PF 1022G substance from the resultant culture of the PF 1022 strain and then purifying and isolating these substances, respectively. Further, we have examined physico-chemical properties of PF 1022G substance, and we have determined the chemical structural formula thereof and thus confirmed PF 1022G substance to be a novel cyclic depsipeptide.
  • PF 1022F substance is a cyclic depsipeptide represented by the following formula (B): ##STR3## wherein Me stands for methyl group and Me has the same meaning in the following descriptions given hereinafter.
  • PF 1022H substance is a cyclic depsipeptide represented by the following formula (C): ##STR4##
  • the novel PF 1022G substance now obtained by the present inventors is a cyclic depsipeptide represented by the following formula (D): ##STR5##
  • PF 1022 strain deposited under FERM BP-2671
  • PF 1022 strain is such a fungus which does not cause any conidium formation.
  • PF 1022 strain is considered surely to belong to the family Xylariaceae and now is presumed to be close to the genus Xylaria or the genus Rosellinia falling under this Xylariaceal family.
  • PF 1022 strain is regarded presumably or tentatively to be a strain belonging to the genus Xylaria or the genus Rosellinia.
  • a process for the preparation of PF 1022F substance, PF 1022G substance and PF 1022H substance which are represented by the following general formula (I) ##STR6## wherein both of R 1 and R 2 each denote methyl group for PF 1022F substance, but R 1 denotes methyl group and R 2 denotes p-hydroxybenzyl group for PF 1022G substance, and both of R 1 and R 2 each denote p-hydroxybenzyl group for PF 1022H substance, which process comprises cultivating a fungal strain capable of producing PF 1022F substance, PF 1022G substance and PF 1022H substance and belonging to the genus Xylaria or the genus Rosellinia, in a culture medium containing carbon and nitrogen sources, thereby producing and accumulating PF 1022F substance, PF 1022G substance and PF 1022H substance in the resulting culture, recovering PF 1022F substance, PF 1022G substance and
  • the fungal strain capable of producing PF 1022F substance, PF 1022G substance and PF 1022H substance which is to be used in the process of the first aspect of this invention, may be any of those strains which are capable of producing PF 1022F substance, PF 1022G substance and PF 1022H substance, as long as it belongs to the genus Xylaria or the genus Rosellinia of the family Xylariaceae.
  • One preferred example of the fungal strains capable of producing PF 1022F substance, PF 1022G substance and PF 1022H substance is the aforesaid PF 1022 strain which is an asporous imperfect fungus and which was isolated from a vegetative sample collected in Ibaraki Prefecture, Japan.
  • the PF 1022 strain has been deposited at National Institute of Bioscience and Human-Technology, Agency of Industrial Science & Technology (located at No.1-3, 1-chome, Higashi, Tsukuba-shi, Ibaraki Prefecture, Japan) since Jan. 24, 1989 under the access number FERM P-10504, and then since Dec. 4, 1989 under the access number FERM BP-2671 in terms of the Budapest Treaty.
  • the PE 1022 strain is labile in its properties.
  • the PF 1022 strain itself, or any mutant as derived from this strain, phenotypic conjugation (spontaneously generated or artificially induced), or genetic recombinant of said strain may be used in practicing the process of this invention, if it can produce the PE 1022F substance, PE 1022G substance and PE 1022H substance.
  • a fungal strain capable of producing PF 1022F substance, PE 1022G substance and PE 1022H substance is cultivated according to the following cultivation procedure.
  • the above-mentioned PE 1022F substance, PE 1022G substance and PE 1022H substance-producing strain is cultivated in a culture medium containing such ordinary carbon source and nitrogen source which can be generally utilized as nutrients by ordinary Microorganisms.
  • Such nutrients there may be used those nutrients which are known to have been utilized for the cultivation of fungi.
  • the carbon sources are usable the ordinarily utilized carbon sources such as glucose, sucrose, starch syrup, dextrin, starch, glycerol, molasses, animal oils, vegetable oils, and the like.
  • the nitrogen sources are the ordinarily utilized nitrogen sources such as soybean flour, wheat germ, corn steep liquor, cotton seed oil, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, and the like.
  • Inorganic salts capable of producing potassium, calcium, magnesium, cobalt, chloride, phosphate, sulfate and other ions may effectively be added to the culture medium, as necessary.
  • organic and inorganic substances capable of promoting the growth of the fungal strain and thus promoting the production of PF 1022F substance, PF 1022G substance and PF 1022H substance may also be added in an appropriate amount.
  • a cultivation method carried out under aerobic conditions is suitable, and particularly the cultivation method under submerged conditions is most suitable.
  • the temperature range of 15 ⁇ 30° C. is suitable for the cultivation, but optimally the cultivation may be effected at a temperature of about 26° C. in most instances.
  • the production of PF 1022F substance, PF 1022G substance and PF 1022H substance will usually arrive at a maximum accumulation of these substances in 2 to 10 days, although the incubation period required therefor may vary depending upon the composition of the culture medium and the cultivation conditions employed.
  • the cultivation step is discontinued.
  • the resulting culture is then separated by filtration or by a centrifugal operation to give solid portion including the cultured cells and other solid materials and to give the broth filtrate.
  • the filtration operation may be effected using a filtering aid such as diatomaceous earth, etc.
  • the recovery of PF 1022F substance, PF 1022G substance and PF 1022H substance as produced by the above cultivation of the microbial strain may be effected in the following manner.
  • the recovery of PF 1022F substance, PF 1022G substance and PF 1022H substance from the resultant culture may generally be carried out by ordinary procedure of separation with utilizing its physicochemical characteristics, for example, by solvent extraction or adsorption, ion-exchange resin treatment, partition column chromatography, gel filtration, dialysis, precipitation and so on, either alone or in an appropriate combination.
  • PF 1022F substance, PF 1022G substance and PF 1022H substance are insoluble in water, these substances exist mainly in the cultured cells rather than in the broth filtrate.
  • PF 1022F, G and H substances may be extracted from the cultured cells with an organic solvent or an aqueous organic solvent, for example, methanol or ethyl acetate, or acetone-water, acetonitrile-water, etc.
  • an organic solvent or an aqueous organic solvent for example, methanol or ethyl acetate, or acetone-water, acetonitrile-water, etc.
  • a chromatographic method with silica gel e.g.
  • Wako gel C-200 produced by Wako Junyaku K.K.
  • alumina or the like as an adsorbent
  • a gel filtration agent such as Sephadex LH-20 (a product of Pharmacia Co.), Toyopal HW-40 (a product of Toso Co., Ltd.).
  • crystallization of each of PF 1022 F substance, PF 1022G substance or PF 1022H substance may be effected from a single solvent (e.g. methanol, ethyl acetate, etc.) or a mixed solvent (e.g. methanol-water, ethyl acetate-n-hexane, diethylether-n-hexane, etc.).
  • PF 1022F substance, PF 1022G substance and PF 1022H substance may be obtained separately from each other at a high purity.
  • the process according to the first aspect of this invention includes the steps of separating from the resulting culture the cultured cells of the strain capable of producing PF 1022F substance, PF 1022G substance and PF 1022H substance, extracting the cultured cells so separated with an organic solvent or an aqueous organic solvent to obtain an extract containing PF 1022F substance, PF 1022G substance and PF 1022H substance, concentrating said extract to allow crystals comprising PF 1022G substance and PF 1022H substance to precipitate therein, filtering the resulting concentrated solution to separate said crystals and the filtrate containing PF 1022F substance, and isolating PF 1022G substance and PF 1022H substance, respectively, from the crystals, and also isolating PF 1022F substance from said filtrate.
  • the present inventors have now found that when the cultivation of PF 1022 strain above-mentioned used as the strain capable of producing the PF 1022F substance, PF 1022G substance and PF 1022 is conducted in such a manner that the cultivation of the PF 1022 strain is carried out in a culture medium containing the ordinary carbon and nitrogen sources and in the presence of p-hydroxyphenyllactic acid or its sodium or potassium salt as added positively in an amount of 0.1 ⁇ 5% by weight based on the weight of the culture medium, it results in that the concentration of PF 1022H substance as produced in said culture medium can be increased as high as about 3 times or more, as compared with such a case when the cultivation of said strain is carried out in the absence of the added p-hydroxyphenyllactic acid, and it has also been found that PF 1022H substance can then be recovered from the resultant culture so formed in an improved yield. In this case, there exists a possibility of increasing the yield of PF 1022G substance, too.
  • a process for the preparation of PF 1022F substance, PF 1022G substance and PF 1022H substance with a highly efficient production of PF 1022H substance comprises cultivating the PF 1022 strain (deposited under FERM BP-2671) as the fungal strain capable of producing PF 1022F substance, PF 1022G substance and PF 1022H substance, in a culture medium containing carbon and nitrogen sources and in the presence of p-hydroxyphenyllactic acid or sodium or potassium salt thereof as added to the culture medium in an amount of 0.1 ⁇ 5% based on the weight of the culture medium, at a cultivation temperature of 15 ⁇ 30° C., continuing the cultivation of the PF 1022 strain until the concentration of PF 1022H substance arrives at or near its maximum in the resulting culture, thereby producing and accumulating in the resulting culture PF 1022F substance, PF 1022G substance and PF 1022H substance along with PF 1022A substance, PF 1022B
  • the cultivation of the PF 1022 strain and the recovery of PF 1022F, G and H substances from the culture can be carried out in the same manner as in the process according to the first aspect of this invention.
  • this process includes the steps of separating the cultured cells of PF 1022 strain from the resulting culture of the PF 1022 strain, extracting the so separated, cultured cells with methanol to obtain the methanolic solution containing PF 1022F substance, PF 1022G substance and PF 1022H substance together with PF 1022A substance, PF 1022B substance, PF 1022C substance, PF 1022D substance and PF 1022E substance, then distilling off the methanol from said methanolic solution to give a concentrated solution, stirring the concentrated solution at room temperature or lower to allow crystals comprising a mixture of PF 1022A substance, PF 1022B substance, PF 1022C substance, PF 1022D substance and PF 1022E substance as well as PF 1022G substance and PF 1022H substance to precipitate, filtering off the crystals from the mother liquor containing PF 1022F substance remaining dissolved therein, subsequently recovering PF 1022
  • a novel cyclic depsipeptide which is PF 1022G substance represented by the following formula: ##STR7##
  • PF 1022G substance is in the form of colorless crystalline powder melting at 138.1 ⁇ 139.4° C. which has physico-chemical properties described in detail after the Examples 1 ⁇ 2 given hereinafter.
  • PF 1022G substance possesses an anthelmintic activity against fowl roundworms.
  • PF 1022F, G and H substances which are produced according to this invention, may be utilized as a starting material for the syntheses of anthelmintically active cyclic depsipeptide derivatives.
  • PF 1022G substance and PF 1022H substance each may be utilized for the preparation of various other cyclic depsipeptide derivatives by treating the hydroxyl group on the benzyl group of PF 1022G or H substance through chemical modification or conversion with using such chemical reactions which are applicable to the normally phenolic hydroxyl group.
  • PF 1022G substance may be used as an intermediate for the syntheses of the compounds disclosed in the specification of WO95/07272 mentioned hereinbefore
  • PF 1022H substance may be used as an intermediate for the syntheses of the compounds disclosed in the specification of WO93/19053 or WO97/11064.
  • the processes of this invention are of industrially high value in that these processes can provide by the convenient procedure PF 1022F ⁇ H substances which are useful as intermediates for synthetic production of other cyclic depsipeptide derivatives by the chemical conversion.
  • FIG. 1 shows a proton nuclear magnetic resonance spectrum of PF 1022F substance as determined in a deutero-chloroform solution at 270 MHz, where the abscissa represents chemical shift (ppm: ⁇ ).
  • FIG. 2 shows a proton nuclear magnetic resonance spectrum of PF 1022G substance as determined in a deutero-chloroform solution at 270 MHz, where the abscissa represents chemical shift (ppm: ⁇ )
  • FIG. 3 shows a proton nuclear magnetic resonance spectrum of PF 1022H substance as determined in a mixed solution of deutero-chloroform-deutero-methanol at 270 MHz, where the abscissa represents chemical shift (ppm: ⁇ )
  • a culture medium comprising 2.0% soluble starch, 1.0% glucose, 0.5% polypeptone, 0.6% wheat germ, 0.3% yeast extract, 0.2% soybean cake, 0.2% calcium carbonate and the balance water was used as the seed culture medium.
  • medium comprising 2.0% glucose, 1.0% starch, 0.8% wheat germ, 1.3% soybean cake, 0.38% meat extract, 0.13% sodium chloride, 0.15% calcium carbonate and the balance water. All these media were used by adjusting the pH to 7.0 before sterilization.
  • the seed culture medium above-mentioned was placed in 40 ml-portions into 200 ml-capacity Erlenmeyer flasks.
  • the seed culture media in the flasks were sterilized at 120° C. for 15 minutes and then inoculated with 2 ⁇ 3 loopfuls of a slant culture of the PF 1022 strain (deposited under FERM BP-2671) per flask. Shake-cultivation was conducted at 26° C. for 48 hours to give a primary seed culture.
  • the seed culture medium of the same composition as above was placed in 500 ml-portions into 2 liter-capacity Erlenmeyer flasks, sterilized at 120° C.
  • the seed culture medium as above 500 liters was charged into a 1 liter capacity-tank fermenter, sterilized at 120° C. for 25 minutes and then inoculated with the content of the five flasks containing the said primary seed culture and subsequently cultivated under stirring at 26° C. for 48 hours to give a tank seed culture.
  • the production culture medium as above (5 kilo-liters) was charged into a 10 kilo-liter capacity-tank fermenter and sterilized at 120° C. for 25 minutes.
  • the production culture medium so sterilized was inoculated with 0.5 kilo-liters of the tank seed culture obtained in the above and then cultivated at 26° C. for 7 days with aeration and under stirring.
  • the culture broth so obtained was filtered with the addition of diatomaceous earth as a filtering aid, to yield the cultured cells of the PF 1022 strain (about 2700 kg).
  • methanol (14.4 kilo-liters) was added to the cultured cells obtained as above (about 2700 kg), and the resultant mixture was stirred for 3 hours. Thereafter, the cells were filtered off to yield the methanolic extract from the cells. The methanol was distilled off from the methanolic extract under a reduced pressure to give a concentrated solution (960 liters). The concentrated solution was stirred at room temperature for a whole day and one night, and the resulting crystals (primary crystals) as deposited were collected by filtration.
  • the primary crystals (about 60 kg) so obtained comprised a mixture of PF 1022A substance, PF 1022B substance, PF 1022C substance, PF 1022D substance, PF 1022E substance, PF 1022F substance, PF 1022G substance, PF 1022H substance and other cell constituents.
  • the filtrate also contained the above-mentioned PF 1022A substance, PF 1022B substance, PF 1022C substance, PF 1022D substance, PF 1022E substance, PF 1022F substance, PF 1022G substance, PF 1022H substance and other cell constituents, but the proportions of these constituents present in the filtrate were different from those of the primary crystals, while the proportion of PF 1022F substance is relatively higher in the filtrate.
  • the primary mother liquor obtained was stored and used later for the recovery of PF 1022F substance therefrom.
  • the primary crystals as above were washed twice with n-heptane (200 liters).
  • the crystals left after the washing were dissolved in methanol (500 liters), to which solution was added activated carbon (12 kg).
  • the resultant mixture was stirred for 1 hour.
  • the resulting filtrate was concentrated under reduced pressure to a volume of 170 liters.
  • the concentrated solution so obtained was stirred at room temperature for 24 hours, and the crystals so precipitated (the secondary crystals) were collected by filtration.
  • the secondary crystals (about 35 kg) comprised a mixture of the PF 1022A substance as a main constituent along with PF 1022C substance, PF 1022D substance, PF 1022E substance and PF 1022G substance.
  • the secondary crystals were washed twice with n-heptane (each 240 liters), then dissolved in methanol (350 liters) and then the methonolic solution was concentrated under a reduced pressure to a volume of 100 liters.
  • the concentrated solution obtained was stirred at room temperature for 24 hours and the crystals thus obtained (the tertiary crystals) was collected by filtration.
  • the tertiary crystals so obtained (about 31 kg) contained PF 1022A substance in a high purity.
  • the crystals so obtained comprised a mixture of the PF 1022A substance, PF 1022B substance, PF 1022C substance, PF 1022D substance, PF 1022E substance, PF 1022G substance and PF 1022H substance.
  • a half amount (4.1 kg) of the crystals so obtained was dissolved in chloroform (10 liters) and the resulting chloroform solution was subjected to a silica gel column chromatography (Wako gel C-300, 20 kg) .
  • the silica gel column was first eluted with a developing solvent (380 liters) consisting of chloroform-ethyl acetate (6:1) and then eluted with a developing solvent (140 liters) consisting of chloroform-ethyl acetate (1:1).
  • Fractions Nos. 8 ⁇ 10 of the eluate contained PF 1022E substance, PF 1022G substance and PF 1022H substance as main constituents and were combined together and then concentrated under reduced pressure.
  • the powder thus obtained (158 g) comprised a mixture of PF 1022E substance, PF 1022G substance and PF 1022H substance.
  • the powder (158 g) was crystallized from ethyl acetate (800 ml) and the crystals was collected by filtration. A portion (2.5 g) of the crystals so obtained (121.6 g) was dissolved in toluene (10 ml), and the solution in toluene was subjected to a silica gel column chromatography (Wako gel C-200, 100 g).
  • the stored filtrate (the mother liquor of the primary crystals) remaining from the filtering operation of the primary crystals above was concentrated to dryness under a reduced pressure to give a powder.
  • a portion (2.35 kg) of said powder was dissolved in chloroform (10 liters) and the resultant solution was subjected to a silica gel column chromatography (Wako gel C-300, 20 kg).
  • the silica gel column was first eluted with a developing solvent (420 liters) consisting of chloroform-ethyl acetate (6:1), and then eluted with a developing solvent (120 liters) consisting of chloroform-ethyl acetate (1:1).
  • Fraction No.10 of the eluate contained PF 1022E substance, PF 1022F substance, PF 1022G substance and other substances so eluted, and the fraction No.10 was concentrated under a reduced pressure.
  • the powder (45.0 g) so obtained was subjected to a silica gel column chromatography (Wako gel C-300, 550 g). Elution was effected with a developing solvent (4 liters) consisting of chloroform-ethyl acetate (3:1) and the eluate was collected in 500 ml-fractions. Fractions Nos.3 -7 containing the PF 1022F substance were combined and then concentrated under reduced pressure, to afford a powder (9.19 g) comprising PF 1022E substance, PF 1022F substance and other substances.
  • This powder was dissolved in a 60% aqueous acetonitrile, and the resulting solution was subjected to a reversed phase column chromatography (Cosmosil 75C180PN, 100 g). The elution was effected first with a 60% aqueous acetonitrile (500 ml), then with a 70% aqueous acetonitrile (400 ml) and finally with an 80% aqueous acetonitrile (900 ml). The eluates obtained were collected in 200 ml-fractions. Eluate fractions Nos.2 ⁇ 8 containing PF 1022F substance were combined and then concentrated under reduced pressure, to afford a crude powder (6.53 g) of PE 1022F substance.
  • the seed culture medium (50 ml) having the same composition as described in Example 1 above was placed into each of 250 ml-Erlenmeyer flasks.
  • the flasks, after sterilizing at 120° C. for 15 minutes, were inoculated with 2 ⁇ 3 loopfuls of a slant culture of the PP 1022 strain and then incubated at 26° C. for 3 days.
  • the resulting culture broth was separated into two portions. To one portion of said two portions was added p-hydroxyphenyllactic acid in an amount of 0.3% by weight based on the weight of the culture medium present in said one portion. Both the two portions were separately further incubated at 26° C. for 3 days.
  • the resulting cultured cells obtained from the incubated two portions of the culture broth were extracted with methanol in a manner similar to the procedure disclosed in Example 1(b).
  • the concentration ( ⁇ g/ml) of PF 1022H substance in the resulting two methanolic extracts was assayed by a liquid column chromatography (column: a product of GL Science Co., Inertsil ODS-2: 4.6 mm ⁇ 250 mm; at temperature of 40° C.; flow rate of 1 ml/min.; detecting UV at 210 nm, mobile phase consisting of 80% acetonitrile +0.1% trifluoroacetic acid; at retention time of 5.0 minutes).
  • the seed culture medium (50 ml) having the same composition as described in Example 1(a) was placed into each of one hundred of 250 ml-Erlenmeyer flasks.
  • the culture medium in the one hundred (100) of the flasks above were sterilized at 120° C. for 15 minutes. Thereafter, the seed culture medium of each of the 100 flasks (total volume of the seed medium: 5 liters) was inoculated with 2 ⁇ 3 loopfuls of the PF 1022 strain (deposited under FERM BP-2671) and then incubated at 26° C. for 3 days.
  • PF 1022 strain was then continued at 26° C. for further 3 days under aeration and stirring. After the completion of the cultivation, the resulting culture broth was filtered with aid of diatomaceous earth as a filtering aid, thus to obtain the cultured cells of the PF 1022 strain (about 2.5 kg).
  • FIG. 1 1 H-nuclear magnetic resonance spectrum of PF 1022F substance as determined in deutero-chloroform at 270 MHz using TMS (tetramethylsilane) as internal standard.
  • Rf value is 0.34, when use is made of an eluent of chloroform-ethyl acetate (1:2) on silica gel plate 60F254 (thickness 0.25 mm, a product of Merck & Co.).
  • FIG. 2 1 H-nuclear magnetic resonance spectrum of PF 1022G substance as determined in deutero-chloroform at 270 MHz using TMS (tetramethylsilane) as internal standard.
  • Rf value is 0.32, when use is made of an eluent of chloroform-ethyl acetate (1:2) on silica gel plate 60F254 (thickness 0.25 mm, a product of Merck & Co.).
  • FIG. 3 1 H-nuclear magnetic resonance spectrum of PF 1022H substance as determined in a mixed solvent of deutero-chloroform-deutero-methanol at 270 MHz using TMS (tetramethylsilane) as internal standard.
  • Rf value is 0.25, when use is made of an eluent of chloroform-ethyl acetate (1:2) on silica gel plate 60F254 (thickness 0.25 mm, a product of Merck & Co.).
  • PF 1022F substance and PF 1022H substance as well as a novel cyclic depsipeptide, PF 1022G substance, have now been produced in a convenient way by the processes according to this invention, which comprise cultivating a fungal strain capable of producing PF 1022F substance, PF 1022G substance and PF 1022H substance, which strain belongs to the genus Xylaria or the genus Rosellinia in the family Xylariaceae.
  • the PF 1022F substance, PF 1022G substance and PF 1022H substance thus produced are the cyclic depsipeptides having anthelmintic activities and also are useful as starting materials to be used for the chemical syntheses of such known or novel derivatives of PF 1022 substance which have higher anthelmintic activities.

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2018166899A1 (en) 2017-03-14 2018-09-20 Acidophil Ltd Methods for production of pf1022a derivatives
US10623012B2 (en) 2018-05-10 2020-04-14 Advanced Energy Industries, Inc. Precision digital to analog conversion in the presence of variable and uncertain fractional bit contributions

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DE19545639A1 (de) * 1995-12-07 1997-06-12 Bayer Ag Verfahren zur Herstellung von substituierten Arylmilchsäure-haltigen Cyclodepsipeptiden mit 24 Ringatomen
JP4156235B2 (ja) 1999-09-29 2008-09-24 明治製菓株式会社 官能基により修飾された二次代謝産物を生産する形質転換体および新規生合成遺伝子
CN101591618B (zh) * 2008-05-28 2011-07-06 中国科学院微生物研究所 一种细小炭角菌菌株及其液体发酵培养方法和应用
RU2011129395A (ru) * 2008-12-16 2013-01-27 Байер Энимэл Хельс ГмбХ Способ получения оптически активных циклических депсипептидов с 24 атомами в цикле, содержащих молочную кислоту и фенилмолочную кислоту, с помощью грибковых штаммов рода rosellinia, а также других родов из семейства ксиляриевые
AU2010329998B2 (en) 2009-12-11 2015-05-21 Bayer Intellectual Property Gmbh Novel 24-membered cyclooctadepsipeptides from fungal strains and their use as anthelmintics or endoparasiticides
CN107217007B (zh) * 2016-03-22 2021-01-29 上海医药工业研究院 一种生产pf1022a的发酵培养基及发酵方法
CN106749569B (zh) * 2017-03-03 2021-10-15 重庆乾泰生物医药有限公司 一种pf1022a的分离纯化方法
CN108570016B (zh) * 2017-03-10 2021-11-26 上海医药工业研究院 一种pf1022a分离纯化的方法
CN109880746B (zh) * 2017-12-06 2023-10-20 海正药业(杭州)有限公司 一种座坚壳属真菌菌株及其应用

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EP0685469A4 (en) * 1993-02-19 1996-04-03 Meiji Seika Kaisha DERIVATIVE OF PF 1022 USED AS A CYCLIC DEPSIPEPTIDE.
EP0780468A4 (en) * 1995-06-22 2000-07-19 Meiji Seika Kaisha TRANSFORMANT WHICH PRODUCES THE SUBSTANCE PF1022 AND METHOD FOR TRANSFORMING MICROORGANISMS IN THE CLASS OF THE HYPOMYCETES
DK0903347T3 (da) * 1995-09-22 2005-11-21 Bayer Ag Hidtil ukendte cyclodepsipeptid PF1022-derivater
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018166899A1 (en) 2017-03-14 2018-09-20 Acidophil Ltd Methods for production of pf1022a derivatives
US10623012B2 (en) 2018-05-10 2020-04-14 Advanced Energy Industries, Inc. Precision digital to analog conversion in the presence of variable and uncertain fractional bit contributions
US10944416B2 (en) 2018-05-10 2021-03-09 Advanced Energy Industries, Inc. Precision digital to analog conversion in the presence of variable and uncertain fractional bit contributions
US11368163B2 (en) 2018-05-10 2022-06-21 Advanced Energy Industries, Inc. Precision digital to analog conversion in the presence of variable and uncertain fractional bit contributions

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KR20000029822A (ko) 2000-05-25
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US6146853A (en) 2000-11-14
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